Methodologies for specific gene targeting or precise genome editing are of great importance to functional characterization of plant or animal genes. In recent years, sequence-specific nucleases have been developed to increase the efficiency of gene targeting or genome editing in plant, animal, or microbial systems.
Recently, a new genome editing tool has been developed in microbial and mammalian systems based on the cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease system. The CRISPR-associated nuclease is part of adaptive immunity in bacteria and archaea. The Cas9 endonuclease, a component of Streptococcus pyogenes type II CRISPR/Cas system, forms a complex with two short RNA molecules called CRISPR RNA (crRNA) and transactivating crRNA (transcrRNA), which guide the nuclease to cleave non-self DNA on both strands at a specific site. The crRNA-transcrRNA heteroduplex could be replaced by one chimeric RNA (so-called guide RNA (gRNA)), which can then be programmed to targeted specific sites. The minimal constrains to program gRNA-Cas9 is at least 15-base-pairing between engineered 5′-RNA and targeted DNA without mismatch, and an NGG motif (so-called protospacer adjacent motif or PAM) follows the base-pairing region in the targeted DNA sequence. Generally, 15-22 nt in the 5′-end of the gRNA region is used to direct Cas9 nuclease to generate DSBs at the specific site. The CRISPR/Cas system has been demonstrated for genome editing in human, mice, zebrafish, yeast and bacteria. Distinct from animal, yeast, or bacterial cells to which recombinant molecules (DNA, RNA or protein) could be directly transformed for Cas9-mediated genome editing, recombinant plasmid DNA is typically delivered into plant cells via the Agrobacterium-mediate transformation, biolistic bombardment, or protoplast transformation due to the presence of cell wall. Thus, specialized molecular tools and methods need to be created to facilitate the construction and delivery of plasmid DNAs as well as efficient expression of Cas9 and gRNAs for genome editing in, plant or animal cells. Compositions and methods for making and using CRISPR-Cas systems are described in U.S. Pat. No. 8,697,359, entitled “CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS,” which is incorporated herein in its entirety.
In principle, multiplex genetic manipulation could be achieved by expressing multiple gRNAs with Cas9 (or Cas9 derived effectors) for corresponding target sites. Due to limitations in delivery method and capacity of current gRNA expressing devices, however, simultaneously producing numerous gRNAs in plant, animal, or microbial organisms is still a challenge.
It is an objective of the present invention to provide a strategy to use a cell's endogenous tRNA processing system as an efficient and precise approach to produce numerous gRNAs from a single polynucleotide construct/synthetic gene, thereby boosting the multiplex editing capability of CRISPR/Cas9 or other RNA mediated genetic manipulation tools for genome engineering.
In another aspect, the present invention relates to a polynucleotide construct, expression cassette, vector or recipient modified cell comprising a nucleic acid as described, supra.
It is a further objective, feature or advantage of the present invention to provide compositions and methods for manipulating multiple target genes, or multiple positions within a single target gene with introduction of a single expression cassette that includes sequences to utilize the tRNA system in the recipient cell.
Additional objectives, features and advantages may become obvious based on the disclosure contained herein.